Effect of itraconazole on Staphylococcus aureus biofilm and extracellular vesicles formation.

Staphylococcus aureus is a leading cause of a wide range of clinical chronic infections mainly due to the establishment of a biofilm. Biofilm, a population of bacteria within a self-produced matrix of extracellular polymeric substance, decreases the susceptibility to antibiotics, immune defenses and contributes to antimicrobial resistance. To date antibiotic combination has been considered a strategy to combat S. aureus infection, but this approach does not solves the main pharmacokinetic problem caused by biofilms, consisting in insufficient drug penetration within the structure. Therefore, new antimicrobial agents that could overcome this resistance need to be discovered. Fighting staphylococcal resistance and biofilm formation is an important goal of the pharmaceutical research. Some fungicide has been observed to have antibacterial effect. anyway their use as antibiotics on S.aureus has been poorly studied. The aim of this work was to investigate the effect of the fungicide itraconazole (IT) on S. aureus biofilm formation and explore by SEM the morphological alteration after treatment. A strong biofilm disaggregation and morphologically different extracellular vesicles (EV) production were observed starting from sublethal IT doses. This suggests that IT resistance phenomena on the part of S. aureus are more difficult to establish respect other antibiotics. The adjuvant properties of IT could be used to combat bacterial biofilm and/or to improve antibiotic treatment. Moreover, because the production of EV represents a secretory pathway involved in intercellular communication shared to mammalian cells, fungi, and bacteria, our study is important to increase information that can be generalized to higher organisms.

Expression profiling of two stress-inducible genes encoding for miraculin-like proteins in citrus plants under insect infestation or salinity stress.

The expression of two genes, namely Mir1 and Mir3 and the abundance of their encoded proteins, the putative miraculin-like proteins, MLP1 and MLP3, showing similarity to the Kunitz family of protease inhibitors, were monitored in the leaves of the citrus variety, 'Clementine' after Tetranychus urticae infestation and elicitor treatments, or in the leaves of three other diploid citrus: 'Willow leaf', 'Cleopatra' mandarins and 'Trifoliate' orange, as well as their respective doubled diploids and the allotetraploid somatic hybrid 'FLHORAG1' under salt stress. RT-PCR and 2-DE indicated that Mir1 and Mir3 and their products were present at low-basal expression in all citrus genotypes. Both genes and products were induced in the 'Clementine' leaves infested by T. urticae, but a contrasting profile was observed under elicitor treatments. Under salt stress, the two genes showed an expression pattern contrasting each other and depending on the genotypes. 'Cleopatra' mandarin, 'Trifoliate' orange and 'FLHORAG1' presented overexpression of Mir3 and MLP3 and decreased levels of Mir1 and MPL1. The opposite behaviour was found in 'Willow leaf' mandarin. The positive correlation of the expression profile of the two genes with that of a gene encoding a putative apoplastic cysteine protease (CysP) might suggest a possible interaction of the respective encoded proteins during the response to biotic stress. Under salt stress, CysP and Mir 1 showed a similar expression pattern but only at transcript level. The possible occurrence of post-translational CysP regulation is discussed.

Expression and characterization of two new alkane-inducible cytochrome P450s from Trichoderma harzianum.

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax).

Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.

Involvement of human p53 in induced intrachromosomal recombination in Saccharomyces cerevisiae.

p53 tumor suppressor protein plays a key role in maintaining genomic integrity and regulating growth control following exposure to DNA-damaging agents. Moreover, it is likely to control genome stability by affecting homologous recombination. p53 can work as a transcription factor in Saccharomyces cerevisiae, therefore this organism represents a good genetic model in which to investigate the molecular mechanism and genetic control of DNA damage-induced recombination. We expressed wild-type human p53 and a mutated form lacking transcriptional activity in S.cerevisiae strain RS112, which carries a synthetic intrachromosomal recombination substrate, and the frequencies of spontaneous and DNA damage-induced homologous recombination were determined. While an increase in intrachromosomal recombination induced by both UV radiation and methylmethane sulphonate (MMS) was observed in yeast cells carrying the void plasmid, p53 expression significantly reduced recombination frequency. The mutated p53 significantly reduced UV-induced recombination but had no effect on MMS-induced recombination. Our results suggest that human p53 inhibits homologous recombination induced by UV and MMS by mechanisms involving stabilization and/or phosphorylation of the protein.

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1.

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.

Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents.

It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.

Genotoxic and mono-oxygenase system effects of the fungicide maneb.

The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIEI P-450 isoform.

Effect of magnetic fields on rodent monooxygenase enzymes.

The effects of 50 Hz, 1.2 mT magnetic fields (MFs) were tested on hepatic monooxygenase enzymes of basal and beta-naphthoflavone-phenobarbital-preinduced rats and mice. An inductive effect on cytochrome P-450 level and on some enzymatic cytochrome P-450-dependent activities was observed in basal mice after MF exposure. Enzymatic activities in preinduced mice and rats were reduced by MFs, the degree of reduction depending on the enzyme. A specific inhibitory effect was determined in some of the assayed activities and in the relative peculiar P-450 isoforms detected by Western blot analysis.

Induction of cytochrome P-450 by 5-methoxypsoralen in the yeast Saccharomyces cerevisiae.

Yeast cells (D7 strain) incubated in the presence of 5-methoxypsoralen (5-MOP) increase the activity of the monooxygenase system cytochrome P-450 dependent (cytochrome P-450 level and 7-ethoxycoumarin-O-diethylase activity). Northern analysis of cytochrome P-450 specific RNA shows that 5-MOP treatments induce an increase in mRNA. The induction of cytochrome P-450 appears to occur at the transcriptional level. The capacity of 5-MOP to induce the cytochrome P-450 system in eukaryotic cells, in which it is known to be involved in the metabolism of the psoralen, may decrease the availability of the compound for photo-induced genotoxic reactions, which may explain the good tolerance in patients.

Study of spermine and spermidine effects on Saccharomyces cerevisiae. Polyamine production in different growth conditions and in the presence of interleukin-2.

The role of polyamines during cell growth is still uncertain. Yeast cells possess an amine pathway similar to that described for animal cells. We studied the relationship between growth and polyamine production in yeast cells of Saccharomyces cerevisiae grown under various conditions. Polyamines were determined in homogenates of yeast cells by a rapid enzymatic assay, confirmed by thin-layer chromatography. Polyamine production is dependent on the growth conditions. The effects of spermine, spermidine, and interleukin-2 on yeast cells were investigated. Exogenous polyamines affected the endogenous pathway, and interleukin-2 treatment increase yeast growth and polyamine production.

Inhibition of yeast cytochrome P-450 by ammonium metavanadate.

Incubation of diploid D7 strain cells of Saccharomyces cerevisiae (grown in 20% glucose) in the presence of ammonium metavanadate (AMV) led to a decrease in the cytochrome P-450-dependent monooxygenase system (cytochrome P-450 level and 7-ethoxycoumarin O-deethylase). The electrophoretic analysis of microsomal fractions of yeast cells treated with metavanadate revealed a decrease in the intensity of the bands corresponding to a M(r) in the range of 51,000-58,000 Da compared with those observed in controls, i.e., cells grown in 20% glucose. Analysis of the cytochrome P-450 transcript showed that AMV treatment reduced the mRNA level. Our results suggest that AMV inhibits the yeast cytochrome P-450 system by acting at both the pre- and post-transcriptional levels.

Expression of cytochrome P450 in yeast after different chemical treatments.

In Saccharomyces cerevisiae a number of chemical agents induce synthesis of cytochrome P450. A cytochrome P450 gene has been well characterized in this yeast: CYP51, which codes for a constitutive enzyme involved in the 14 alpha-demethylation of lanosterol, a key step in the biosynthesis of ergosterol. In this work, we have analysed the level of transcription of the CYP51 gene in correlation with cytochrome P450 enzymatic activity after treatment with several chemical agents known to interact with cytochrome P450. Using as a probe a DNA fragment whose identity to the CYP51 gene was established by sequence analysis and mapping on chromosome VIII, a unique RNA species was observed in all treatment samples. The increased level found for this transcript in cells treated with ethanol, 20% glucose, phenobarbital or 5-methoxypsoralen correlates with the levels of induction in cytochrome P450 enzymatic activity measured in cells grown under the same conditions, indicating that induction of cytochrome P450 by these treatments is regulated at the transcriptional level.

Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae.

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

Genotoxicity of vanadium compounds in yeast and cultured mammalian cells.

The ability of vanadium compounds to induce genetic activity was investigated in D7 and D61M strains of Saccharomyces cerevisiae and in Chinese hamster V79 cell line. In our previous work, ammonium metavanadate (pentavalent form, V5) induced mitotic gene conversion and point reverse mutation in the D7 strain of yeast. The genotoxicity was reduced by the presence of S9 fraction, which probably reduced pentavalent vanadium to the tetravalent form. In the present study, vanadyl sulfate (tetravalent form, V4) induced no convertants and revertants in yeast cells harvested from stationary growth phase. With yeast cells from logarithmic growth phase, which contain high levels of cytochrome P-450, a significant increase in genetic effects was observed. Further experiments, performed by treating cells harvested from logarithmic growth phase in the presence of cytochrome P-450 inhibitors, indicated that the monooxygenase system influenced the genotoxicity of metavanadate while the genetic activity of vanadyl remained unaffected. Aneuploidy effect in the D61M strain of Saccharomyces cerevisiae was induced by either V5 or V4, confirming that vanadium compounds are potentially antitubulin agents in eukaryotic cells. Although these compounds are very toxic in V79 cells, no mutagenic effect was observed in the presence or in the absence of S9 fraction.

Vanadium: genetical and biochemical investigations.

Ammonium metavanadate was studied for its ability to induce mitotic gene conversion and reverse point mutation in the D7 strain of Saccharomyces cerevisiae. Metavanadate increased the convertant and revertant frequencies; the highest activity was observed without metabolic activation. This indicated that the S9 hepatic fraction and yeast cells in logarithmic phase (and containing a high level of cytochrome P450) biotransform vanadate, probably reducing it to vanadyl. In addition, the effect of ammonium metavanadate on the hepatic monooxygenase system was studied in mice by measuring the level of cytochrome P450 and determining the activities of aminopyrine N-demethylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase in mouse liver microsomal fraction. The results indicated that this compound reduced mono-oxygenase activity and also the level of cytochrome P450.

Induction of cytochrome P-450 and catalase activity in Saccharomyces cerevisiae by UV and X-ray irradiation. Possible role for cytochrome P-450 in cell protection against oxidative damage.

Cytochrome P-450 was induced both in the diploid wild-type D7 strain and in two isogenic DNA-repair-deficient strains (rad3 and rad56) of Saccharomyces cerevisiae following UV- and X-irradiation. The induction occurred only in logarithmic growth phase cells and it was transient showing a peak 3 h after irradiation. The maximal amount of cytochrome P-450 was directly proportional to the radiation dose applied. Under the same experimental conditions an increase of the catalase activity was also observed, suggesting that activated oxygen species produced by irradiation might be implicated in the induction of both enzymes. The sensitivity to H2O2 of cells containing high cytochrome P-450 levels was enhanced when this enzyme was specifically inhibited by tetrahydrofuran and metyrapone. This supports the hypothesis that cytochrome P-450, as well as catalase, might be involved in cell protection against oxidative damage.

Tetrachloroethane, pentachloroethane, and hexachloroethane: genetic and biochemical studies.

Tetrachloroethane (TTCE), pentachloroethane (PCE), and hexachloroethane (HCE) were tested in diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension test with and without mammalian metabolic activation (S9). TTCE, PCE, and HCE gave positive results on cells harvested from logarithmic growth phase; only PCE induced a significant increase (P less than or equal to .01) of mitotic gene conversion and point reverse mutation on cells from stationary growth phase with metabolic activation (S9). The in vivo effects on cytochrome P450 content (cyt. P450), pentoxyresorufin O-dealkylase (P450-like, class IIB, PROD), and ethoxy-resorufin O-deethylase (P448-like, class IA, EROD) activities were examined in hepatic microsomes from mice 24 h after acute intoxication. All the halogenated hydrocarbons displayed a marked toxic effect as shown by the significant decrease in cyt. P450 levels (maximum of 76% decrease, with TTCE 753.2 mg/kg) and EROD (maximum of 69% decrease, with PCE 925.4 mg/kg), and to a lesser extent in PROD (maximum of 52.4% decrease, with HCE 3150 mg/kg). Although a general decrease of P450 functions was observed, the toxic effects of TTCE and PCE seem to be preferentially related to P448 forms.